There are a variety of expression systems by which a protein or peptide can be recombinantly produced. Expression systems, wherein the protein or peptide is expressed and displayed as a part of the host cell surface, have applications including use as a peptide display library, as inactivated or live vaccines, and as antigens to generate antibodies. A protein or peptide, in a form isolated from an expression system, may be used as immunogens in vaccine formulations for active immunization; and can be used to generate protein-specific and peptide-specific antisera useful for passive immunization, and as reagents for diagnostic assays.
Expression of a peptide, in a recombinant host cell, has met with limited success. Frequently, peptides are poorly expressed in bacteria because intracellularly the peptide may encounter one or more difficulties including insolubility, instability, degradation, or sequestration. If a peptide is successfully recombinantly produced and purified, such isolated peptide may have reduced biological activity compared to the same amino acids which are part of the protein from which the peptide sequence is derived. Such reduced activity is typical because the active domain of the peptide is conformational rather than linear; and thus, in solution, the isolated peptide does not occur in proper conformation for full biological activity, or such proper conformation is only one of many alternative structures of the peptide.
To overcome the difficulties encountered in recombinant expression of peptides, there have been various attempts in the art to express a peptide in a conformation reflective of the conformation of the peptide when expressed as part of the protein from which it is derived. For example, one approach is to express the peptide as part of a fusion protein. Typically, a fusion protein consists of a microbial (e.g. bacterial) polypeptide backbone into which is incorporated an amino acid sequence representing one or more heterologous peptide sequences. A step in fusion protein expression comprises inserting into a gene, encoding the microbial polypeptide, a nucleic acid sequence encoding one or more heterologous peptides. Usually the gene is part of an expression vector, such that when the vector is introduced into a host cell system, the fusion protein is then produced either as remaining host cell-associated, or secreted into the culture medium of the expression system. Examples of microbial polypeptide backbone includes Escherichia coli proteins Lam B (Charbit et al., 1991, J. Bacteriol. 173:262-275), LacZ, trpE, maltose-binding protein, and thioredoxin (U.S. Pat. No. 5,292,646); a fusion protein including a portion of an E. coil or Salmonella lipoprotein with OmpA or Omp C or Omp F or Omp T (U.S. Pat. No. 5,348,867); bacterial flagellin (U.S. Pat. No. 4,801,536); Schistosoma japonicum glutathione-S-transferase; baculovirus polyhedrin (U.S. Pat. No. 4,745,051); and filamentous phage pIII (Scott and Smith, 1990, Science 249:386-390).
In the art of recombinant protein expression, there remains a need for new systems, and new compositions for the production and delivery of biologically active, stable peptides for use in diagnostic and therapeutic applications. One such need involves the delivery of biologically active, stable peptides to the respiratory tract for inducing mucosal immunity to one or more respiratory tract pathogens.